Glycoproteins in membranes have an important role as receptors for the regulation of growth and metabolism. The enzyme nicotinamide adenine dinucleotide glycohydrolase (E.C.3.2.2.5.) was studied in order to utilize a known isolatable membrane glycoprotein as an eventual means for measuring the specific modifications that occur with such proteins during neoplasia. This enzyme was solubilized and isolated from both microsomes and plasma membranes of rat liver homogenates. The compositional analysis of the microsomal enzyme revealed the presence of approximately 12 percent sugar, most of which consisted of neutral hexoses. Multiple activity peaks were observed for the microsomal enzyme by isoelectric focusing. This profile was markedly different from that observed for the plasma membrane enzyme. However, when both of these proteins were treated with an enzyme that removes sialic acid from glycoproteins, a single coincident peak was observed by isoelectric focusing. We conclude from these experiments that the sugar composition of this glycoprotein enzyme is determined by subcellular localization, or vice versa, and that no compositional differences are likely to be observed in the polypeptide backbone of the glycoprotein isolated from either membrane preparation.